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Hi Ed,
Long time no talk!
I started the #Why-o-Why series in Twitter to clarify some basic theoretical cryo-EM principles – in digestible units – to both newcomers and established senior scientists alike!
In #Why-o-Why #13, for example, I emphasise the fact that the FRC/FSC (and its thresholds) are not tied to structural biology and also not tied to what you (want to) see in the results. Judging the quality of a metric by whether you see protein side chains is a methodological mistake! It is obviously inappropriate when you want to use your intuitive side-chain metric to judge the “results-resolution” achieved in an X-ray tomographic reconstruction of a human brain, or in the 3D tomographic reconstruction of a computer chip. How would you react when a medic tells you your 3D reconstruction of a ribosome is rubbish because he/she cannot even see the 2mm-sized kidney stones she/he likes to distinguish in the 3D reconstructions – but in your ribosome map!
All your FSC examples violate this basic principle and thus do not represent appropriate, clean, scientific counter arguments for any FSC/FRC discussion!
Moreover, issues like the alignments of molecular images in the context single-particle cryo-EM analysis also have ABSOLUTELY NOTHING to do with the FRC/FSC criterion for comparing of two images or two 3D densities. Whatever reference bias or symmetry error you chose to impose on the two images / volumes is your own responsibility.
As I put it in an earlier internet discussion: “If you overheat your living room, don’t blame your thermometer!” (But you may want to check the functioning of your thermostat!). This statement in its cryo-EM translation: Don’t blame the FSC curve for your own Bias Blunders.
In your 2016 paper you introduce the FSC as follows:
“However, the definition of resolution in cryo-EM is a much more nebulous entity than in X-ray crystallography, and the most commonly used method of reporting resolution using a plot of the Fourier Shell Correlation (FSC) is fraught with problems”
Hardly a clean neutral introduction of a scientific topic and certainly a statement that is in flagrant contradiction with the facts (see: #Why-o-Why #13). I know… I am not going to convince you after all these decades of “some people” not willing to read - or listen to clean scientific arguments. I don’t even expect a reaction from you! What I am more worried about is the effect of planting wrong ideas in the minds of a new generation of cryo-EM scientists.
So you cryo-EM newcomers out there, do take the time to read some of the basics; and for those not on Twitter I added #Why-o-Why #13, below (@Marin_van_Heel).
Cheers,
Marin
_______________________________________________I had studiously avoided getting involved in this thread which might lead to a long exchange with Marin about the history, significance and ontological status of the FSC, but I will suggest a paper that while dated addresses some of these isssues:
Subramaniam, S., Earl, L.A., Falconieri, V., Milne, J.L., and Egelman, E.H. (2016). Resolution advances in cryo-EM enable application to drug discovery. Curr Opin Struct Biol 41, 194-202.
Abstract
The prospect that the structures of protein assemblies, small and large, can be determined using cryo-electron microscopy (cryo-EM) is beginning to transform the landscape of structural biology and cell biology. Great progress is being made in determining 3D structures of biological assemblies ranging from icosahedral viruses and helical arrays to small membrane proteins and protein complexes. Here, we review recent advances in this field, focusing especially on the emerging use of cryo-EM in mapping the binding of drugs and inhibitors to protein targets, an application that requires structure determination at the highest possible resolutions. We discuss methods used to evaluate the information contained in cryo-EM density maps and consider strengths and weaknesses of approaches currently used to measure map resolution.
We discuss that FSC is NOT a measure of resolution, it is a measure of reproducibility. We give examples for structures with symmetry where one can impose the wrong symmetry and get a better FSC than with the correct symmetry (this is also discussed in Egelman, E.H. 2014. Ambiguities in helical reconstruction. Elife 3.). While the map looks like garbage, the FSC does not care as the garbage is highly reproducible. As to actually looking at the map, I would agree with previous people who have commented or implied that the gold standard in x-ray crystallography has always been the visual appearance and interpretability of the map, and not some single metric.
Regards,
Ed
Edward Egelman
Harrison Distinguished Professor
Department of Biochemistry and Molecular Genetics
University of Virginia
phone: 434-924-8210
fax: 434-924-5069
www.people.virginia.edu/~ehe2n
From: 3dem <[log in to unmask]> on behalf of Pawel Penczek <[log in to unmask]>
Date: Thursday, February 13, 2020 at 8:42 AM
To: 3dem <[log in to unmask]>
Subject: [3dem] Which resolution?
Dear Teige,
I am wondering whether you are familiar with
Resolution measures in molecular electron microscopy.
Penczek PA. Methods Enzymol. 2010.
Citation
Methods Enzymol. 2010;482:73-100. doi: 10.1016/S0076-6879(10)82003-8.
You will find there answers to all questions you asked and much more.
Regards,
Pawel Penczek
Regards,
Pawel
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